The mammalian reproductive system is critically dependent upon pulsatile hormone release patterned and driven by a small population of gonadotropin-releasing hormone (GnRH) neurons. The scattered distribution of GnRH neurons within the brain has made it extremely difficult to investigate the nature and properties of these cells in any selective manner in vivo.

We report here a procedure that enables selective, high-fidelity activation of the GnRH neurons that control gonadotropin secretion in ovariectomized mice. Using this approach, we have been able to define minimal parameters of activation required to generate pulsatile gonadotropin secretion in the blood. This provides critical information for understanding and manipulating the genesis of gonadotropin pulsatility in reproductive biology. The mechanisms responsible for generating the pulsatile release of gonadotropins from the pituitary gland are unknown.

We develop here a methodology in mice for controlling the activity of the gonadotropin-releasing hormone (GnRH) neurons in vivo to establish the minimal parameters of activation required to evoke a pulse of luteinizing hormone (LH) secretion. Injections of Cre-dependent channelrhodopsin (ChR2)-bearing adeno-associated virus into the median eminence of adult GnRH-Cre mice resulted in the selective expression of ChR2 in hypophysiotropic GnRH neurons.

Acute brain slice experiments demonstrated that ChR2-expressing GnRH neurons could be driven to fire with high spike fidelity with blue-light stimulation frequencies up to 40 Hz for periods of seconds and up to 10 Hz for minutes. Anesthetized, ovariectomized mice had optical fibers implanted in the vicinity of GnRH neurons within the rostral preoptic area. Optogenetic activation of GnRH neurons for 30-s to 5-min time periods over a range of different frequencies revealed that 10 Hz stimulation for 2 min was the minimum required to generate a pulse-like increment of LH. The same result was found for optical activation of GnRH projections in the median eminence.

Increases in LH secretion were compared with endogenous LH pulse parameters measured from ovariectomized mice. Driving GnRH neurons to exhibit simultaneous burst firing was ineffective at altering LH secretion. These observations provide an insight into how GnRH neurons generate pulsatile LH secretion in vivo. Reproductive functioning in all mammals is critically dependent upon pulsatile gonadotropin secretion (). Experiments undertaken in the 1980s clearly established that pulsatile luteinizing hormone (LH) and follicle-stimulating hormone secretion were generated by the episodic release of gonadotropin-releasing hormone (GnRH) into the pituitary portal vasculature (–).

However, a quarter of a century since those experiments were performed, the components and mechanisms responsible for this episodic release of GnRH remain unknown and represent one of the most important unanswered questions in reproductive biology (). Key parameters such as the number of GnRH neurons involved in a pulse and their patterns of electrical firing are unknown. Dj Mitsu The Beats Extra Feeding Rar Extractor. An important insight into the dynamics of a GnRH pulse has come from fast portal blood sampling in ovariectomized sheep where each GnRH pulse is reported to approximate a square wave beginning sharply over 2 min, remaining elevated for ∼5 min, and then falling to baseline over the next 3 min (). This allowed speculation that a subgroup of GnRH neurons may fire coordinately for a period of 2–7 min to generate a pulse of GnRH (). Disappointingly, however, direct electrical recordings of adult GnRH neurons in acute brain slices in vitro have provided no clear correlate of pulsatile hormone secretion (, ).